Master molecular rheostat switch for cell signaling

ABSTRACT

Methods of detecting cell signaling disorders associated with PINCH and ILK, including cell proliferative disorders such as breast cancer are disclosed. Methods of treating and diagnosing disease and disorders associated with PINCH and ILK cell signaling are also disclosed.

FIELD OF THE INVENTION

[0001] This invention relates to cellular regulation, and moreparticularly to cell signaling in cell proliferative disorders.

BACKGROUND OF THE INVENTION

[0002] PINCH (Particularly Interesting New Cys-His protein) is aLIM-only protein consisting primarily of five LIM domains. The LIMmotif, recognized in 1990 in the lin-11, isl-1 and mec 3 proteins,specifies a double zinc finger domain which has been shown toparticipate in protein-protein interactions. Within the LIM family PINCHhas the largest number of LIM domains (five), giving rise to ten zincfingers.

[0003] The function of LIM domain proteins as adapters and modifiers inprotein interactions has been reviewed recently. PINCH likely functionsas an adapter protein for signal transduction. Adapter molecules such asPINCH can control the location, assembly and function of signalingnetworks, and may be constitutively-anchored to a particular subcellularlocalization or may be recruited to a signaling site. Because PINCH isassociated with β1 integrin, a protein localized to the plasma membrane,it is reasonable to assume that PINCH functions as an anchoring adapterprotein, targeting signaling components to sites of signal transductionat the cell membrane.

[0004] The PINCH signaling complex also contains the integrin-linkedkinase (ILK), a serine-threonine kinase that associates with thecytoplasmic tails of integrins β1 and β3. ILK is involved inintegrin-mediated signaling as well as in the β-catenin/LEF-1 signalingpathway, participating in the complex signaling interactions that occurat cell-matrix and cell-cell junctions. ILK may function in crosstalkbetween cell-matrix and cell-cell junctions and also with components ofthe Wnt signaling pathway.

[0005] ILK has been shown to have oncogenic properties. ILK-overexpressing cells are tumorigenic in nude mice. The mechanisms by whichILK up regulation leads to a transformed phenotype are as yet poorlyunderstood, but the available information points to effects on thenucleus. ILK over expression leads to up regulation of specificcell-cycle associated proteins and to the translocation of β-cateninfrom the cell membrane to the nucleus where it forms a complex with thetranscription factor LEF-1. Because ILK over expression in culturedepithelial cells leads to enhanced fibronectin matrix assembly (afeature of mesenchymal cells), it is possible that ILK over expressionin epithelial cells is associated with activation of mesenchymal geneexpression.

SUMMARY OF THE INVENTION

[0006] The present invention provides a method of inhibiting a cellsignaling disorder comprising administering to a subject a compositioncontaining an agent which regulates a PINCH polypeptide or PINCHexpression. The method is useful in inhibiting a cell proliferativedisorder.

[0007] In one embodiment the invention provides a method of diagnosing acell proliferative disorder in a subject associated with PINCH bydetermining the level of PINCH in the sample and comparing the level ofPINCH in the sample to the level of PINCH in a standard sample, whereinan elevated level of PINCH is indicative of a cell proliferativedisorder.

[0008] In another embodiment, the invention provides a method ofameliorating a cell proliferative disorder associated with PINCH,comprising treating a subject having the disorder with an agent thatregulates PINCH activity or expression. The agent can be an antibody,polypeptide, antisense molecule or chemical.

[0009] In a further embodiment, the invention provides a method foridentifying a compound which modulates cell proliferation, by contactinga sample containing PINCH polypeptide with a compound suspected ofhaving PINCH modulating activity and detecting an effect on cellproliferation.

[0010] The invention also provides a method for identifying a cellproliferative disorder in a subject comprising, quantifying theexpression of PINCH, ILK, or a combination thereof and correlating thelevel of expression with the presence of a cell proliferative disorder,wherein an elevated level of PINCH is indicative of a cell proliferativedisorder and wherein an elevated level of ILK is indicative of ametastatic cell proliferative disorder.

[0011] In another embodiment, the invention provides a method fordetecting a cell proliferative disorder in a subject, comprising,quantifying expression of polynucleotides encoding PINCH, ILK or acombination thereof wherein the polynucleotide level determines thepresence of the cell proliferative disorder.

[0012] In yet another embodiment, the invention provides a method fordetecting a cell proliferative disorder in a subject comprisingquantifying PINCH polypeptide, ILK polypeptide or a combination thereof,wherein the levels of PINCH and ILK when compared to a standard sampleare indicative of the presence of a cell proliferative disorder.

[0013] In yet another embodiment the invention provides a method ofdiagnosing breast cancer in a subject comprising detecting PINCH incells isolated from the subject, wherein an elevated amount of PINCHwhen compared to a standard sample is indicative of a breast caner.

[0014] Furthermore, the invention provides a method of diagnosing ametastatic breast cancer in a subject comprising detecting PINCH and ILKin cells isolated from the subject, wherein in an elevated amount ofPINCH and ILK compared to a standard sample is indicative of ametastatic breast cancer.

[0015] The invention also provides a method of determining the prognosisof a patient having a cell proliferative disorder comprising determiningthe level of PINCH and ILK in cells of a patient and correlating thelevel with prognosis of the patient.

[0016] The details of one or more embodiments of the invention are setforth in the accompanying drawings and the description below. Otherfeatures, objects, and advantages of the invention will be apparent fromthe description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

[0017]FIG. 1 is a table representing a spectrum of breast cancerdiagnoses and varied expression of known prognostic indicators.

[0018]FIG. 2 depicts the nucleic acid sequence (SEQ ID NO:1) and thecorresponding amino acid sequence (SEQ ID NO:2) of PINCH.

DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention relates to a method of detecting,diagnosing, and treating signal transduction disorders, such as thoserelated to a cell proliferative disorder, neoplasm or cancer using PINCHpolypeptides, antibodies to PINCH polypeptides, and PINCHpolynucleotides, including antisense molecules, ribozymes andcomplementary polynucleotides.

[0020] In its broadest sense, the present invention allows the detectionof any PINCH-associated disorder in any organ, tissue, or cell, wherethe target polynucleotide sequence or polypeptide sequence encodes aPINCH polypeptide or is a PINCH polynucleotide. Thus, the targetpolynucleotide sequence may be, for example, a mutant polynucleotide, arestriction fragment length polymorphism (RFLP), a polynucleotidedeletion, a polynucleotide substitution, or any other mammalian nucleicacid sequence of interest encoding a PINCH polypeptide. Additionally,the present invention allows for the detection of PINCH polypeptides orfragments thereof. The PINCH polypeptides are described in U.S. Pat. No.5,552,529 the disclosure of which is incorporated herein by reference.

[0021] As used herein, the term “nucleic acid,” “polynucleotide,”“oligonucleotide” or “nucleic acid sequence” refers to a polymer ofdeoxyribonucleotides or ribonucleotides, in the form of a separatefragment or as a component of a larger construct. For example, nucleicacids can be assembled from cDNA fragments or from polynucleotides togenerate a synthetic gene which is capable of being expressed in arecombinant transcriptional unit. Oligonucleotide or nucleic acidsequences of the invention include DNA, RNA, and cDNA sequences.

[0022] A “promoter” is a minimal DNA sequence sufficient to directtranscription of a DNA sequence to which it is operably linked. A“promoter” also includes promoter elements sufficient forpromoter-dependent gene expression controllable for cell-type specificexpression, tissue-specific expression, or inducible by external signalsor agents; such elements may be located in the 5′ or 3′ regions of thenative gene.

[0023] The term “operably associated” refers to functional linkagebetween the regulatory (e.g. promoter) sequence and the nucleic acidregulated by the regulatory sequence. The operably linked regulatorysequence controls the expression of the product. The regulatory sequencemay be heterologous to the desired gene sequence.

[0024] A “vector” is any compound or formulation, biological orchemical, that facilitates transformation or transfection of a targetcell with a polynucleotide of interest, for example antisenseoligonucleotides. Exemplary biological vectors include viruses,particularly attenuated and/or replication-deficient viruses. Exemplarychemical vectors include lipid complexes and DNA constructs.

[0025] To “inhibit” or “inhibiting” activity is to reduce that activitya measurable amount, preferably a reduction of at least 30% or more.Where there are multiple different activities that may be inhibited (forexample, antisense molecules that bind polynucleotides encoding PINCH orILK may have the ability to reduce expression of the PINCH or ILKprotein, the reduction of any single activity (with or without the otheractivities) is sufficient to fall within the scope of this definition.

[0026] To “specifically bind” is to preferably hybridize to a particularpolynucleotide species. The specificity of the hybridization can bemodified and determined by standard molecular assays known to thoseskilled in the art.

[0027] A “suppressive-effective” amount is that amount of the construct,for example an antisense construct, administered in an amount sufficientto suppress the expression of the target, e.g., inhibit translation ofmRNA, by at least 75% of the normal expression, and preferably by atleast 90%. The effectiveness of the construct can be determinedphenotypically or by standard Northern blot analysis orimmunohistochemically, for example. Other standard nucleic aciddetection techniques or alternatively immunodiagnostic techniques willbe known to those of skill in the art (e.g., Western or Northwesternblot analysis).

[0028] Diagnostic Techniques

[0029] The invention provides a method for detecting a cell signalingdisorder associated with PINCH or a cell proliferative disorderassociated with PINCH in a tissue of a subject, comprising contacting atarget cellular component suspected of expressing PINCH or having aPINCH associated disorder, with a reagent which binds to the component.The target cell component can be nucleic acid, such as DNA or RNA, orprotein. When the component is nucleic acid, the reagent is apolynucleotide probe or PCR primer. When the cell component is apolypeptide, the reagent is an antibody probe. The probes can bedetectably labeled, for example, with a radioisotope, a fluorescentcompound, a bioluminescent compound, a chemiluminescent compound, ametal chelator, or an enzyme. Those of ordinary skill in the art willknow of other suitable labels for binding to the antibody, or will beable to ascertain such, using routine experimentation.

[0030] For purposes of the invention, an antibody or nucleic acid probespecific for PINCH may be used to detect the presence of PINCHpolypeptide (using antibody) or polynucleotide (using nucleic acidprobe) in biological fluids or tissues. Oligonucleotide primers based onany coding sequence region in the PINCH sequence are useful foramplifying polynucleotides encoding PINCH, for example by PCR. Anyspecimen containing a detectable amount of polynucleotide or antigen canbe used. A preferred sample of this invention is breast tissue.Alternatively, biological fluids such as blood may be used which maycontain cells indicative of a PINCH-associated cell proliferativedisorder. Preferably the subject is human.

[0031] A number of techniques known to those skilled in the art may beused to detect polynucleotides encoding PINCH. For example, RNAseprotection assays may be used if RNA is the polynucleotide obtained fromthe sample. In this procedure, a labeled antisense RNA probe ishybridized to the complementary polynucleotide in the sample. Theremaining unhybridized single-stranded probe is degraded by ribonucleasetreatment. The hybridized, double stranded probe is protected from RNAsedigestion. After an appropriate time, the products of the digestionreaction are collected and analyzed on a gel (see for example Ausubel etal., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, section 4.7.1 (1987)). Asused herein, “RNA probe” refers to a polynucleotide capable ofhybridizing to RNA in a sample of interest. Those skilled in the artwill be able to identify and modify the RNAse protection assay specificto the polynucleotide to be measured, for example, probe specificity maybe altered, hybridization temperatures, quantity of nucleic acid as wellas other variables known to those skilled in the art. Additionally, anumber of commercial kits are available, for example, RiboQuantTMMulti-Probe RNAse Protection Assay System (Pharmingen, Inc., San Diego,Calif.).

[0032] Additionally, detection of a polynucleotide encoding PINCH may beperformed by standard methods such as size fractionating the nucleicacid. Methods of size fractionating the DNA and RNA are well known tothose of skill in the art, such as by gel electrophoresis, includingpolyacrylamide gel electrophoresis (PAGE). For example, the gel may be adenaturing 7 M or 8 M urea-polyacrylamide-formamide gel. Sizefractionating the nucleic acid may also be accomplished bychromatographic methods known to those of skill in the art.

[0033] The detection of polynucleotides may optionally be performed byusing radioactively labeled probes. Any radioactive label may beemployed which provides an adequate signal. Other labels includeligands, which can serve as a specific binding pair member for a labeledligand, and the like. The labeled preparations are used to probe for apolynucleotide by the Southern or Northern hybridization techniques, forexample. Nucleotides obtained from samples are transferred to filtersthat bind polynucleotides. After exposure to the labeled polynucleotideprobe, which will hybridize to nucleotide fragments containing targetnucleic acid sequences, the binding of the radioactive probe to targetnucleic acid fragments is identified by autoradiography (see GeneticEngineering, 1, ed. Robert Williamson, Academic Press (1981), pp.72-81). The particular hybridization technique is not essential to theinvention. Hybridization techniques are well known or easily ascertainedby one of ordinary skill in the art. For example, RNA is separated on agel transferred to nitrocellulose and probed with complementary DNA tothe sequence of interest. The complementary probe may be labeledradioactively, chemically etc. Hybridization of the probe is indicativeof the presence of the polynucleotide of interest. The relativeintensity of a reporter attached to the probe, for example aradionucleotide, can be indicative of the amount of PINCH polynucleotidepresent in the sample. As improvements are made in hybridizationtechniques, they can readily be applied in the method of the invention.

[0034] The polynucleotides encoding a PINCH or ILK polypeptide may beamplified before detecting. The term “amplified” refers to the processof making multiple copies of the nucleic acid from a singlepolynucleotide molecule. The amplification of polynucleotides can becarried out in vitro by biochemical processes known to those of skill inthe art. The amplification agent may be any compound or system that willfunction to accomplish the synthesis of primer extension products,including enzymes. Suitable enzymes for this purpose include, forexample, E. coli DNA polymerase I, Taq polymerase, Klenow fragment of E.coli DNA polymerase I, T4 DNA polymerase, other available DNApolymerases, polymerase muteins, reverse transcriptase, ligase, andother enzymes, including heat-stable enzymes (i.e., those enzymes thatperform primer extension after being subjected to temperaturessufficiently elevated to cause denaturation). Suitable enzymes willfacilitate combination of the nucleotides in the proper manner to formthe primer extension products that are complementary to each mutantnucleotide strand. Generally, the synthesis will be initiated at the 3′end of each primer and proceed in the 5′ direction along the templatestrand, until synthesis terminates, producing molecules of differentlengths. There may be amplification agents, however, that initiatesynthesis at the 5′ end and proceed in the other direction, using thesame process as described above. In any event, the method of theinvention is not to be limited to the embodiments of amplificationdescribed herein.

[0035] One method of in vitro amplification which can be used accordingto this invention is the polymerase chain reaction (PCR) described inU.S. Pat. Nos. 4,683,202 and 4,683,195. The term “polymerase chainreaction” refers to a method for amplifying a DNA base sequence using aheat-stable DNA polymerase and two oligonucleotide primers, onecomplementary to the (+)-strand at one end of the sequence to beamplified and the other complementary to the (−)-strand at the otherend. Because the newly synthesized DNA strands can subsequently serve asadditional templates for the same primer sequences, successive rounds ofprimer annealing, strand elongation, and dissociation produce rapid andhighly specific amplification of the desired sequence. The polymerasechain reaction is used to detect the presence of polynucleotidesencoding cytokines in the sample. Many polymerase chain methods areknown to those of skill in the art and may be used in the method of theinvention. For example, DNA can be subjected to 30 to 35 cycles ofamplification in a thermocycler as follows: 95° C. for 30 sec, 52° to60° C. for 1 min, and 72° C. for 1 min, with a final extension step of72° C. for 5 min. For another example, DNA can be subjected to 35polymerase chain reaction cycles in a thermocycler at a denaturingtemperature of 95° C. for 30 sec, followed by varying annealingtemperatures ranging from 54-58° C. for 1 min, an extension step at 70°C. for 1 min and a final extension step at 70° C.

[0036] The primers for use in amplifying the polynucleotides of theinvention may be prepared using any suitable method, such asconventional phosphotriester and phosphodiester methods or automatedembodiments thereof so long as the primers are capable of hybridizing tothe polynucleotides of interest. One method for synthesizingoligonucleotides on a modified solid support is described in U.S. Pat.No. 4,458,066. The exact length of primer will depend on many factors,including temperature, buffer, and nucleotide composition. The primermust prime the synthesis of extension products in the presence of theinducing agent for amplification.

[0037] Primers used according to the method of the invention arecomplementary to each strand of nucleotide sequence to be amplified. Theterm “complementary” means that the primers must hybridize with theirrespective strands under conditions which allow the agent forpolymerization to function. In other words, the primers that arecomplementary to the flanking sequences hybridize with the flankingsequences and permit amplification of the nucleotide sequence.Preferably, the 3′ terminus of the primer that is extended has perfectlybase paired complementarity with the complementary flanking strand.

[0038] Those of ordinary skill in the art will know of variousamplification methodologies which can also be utilized to increase thecopy number of target nucleic acid. The polynucleotides detected in themethod of the invention can be further evaluated, detected, cloned,sequenced, and the like, either in solution or after binding to a solidsupport, by any method usually applied to the detection of a specificnucleic acid sequence such as another polymerase chain reaction,oligomer restriction (Saiki et al., Bio/Technology 3: 1008-1012 (1985)),allele-specific oligonucleotide (ASO) probe analysis (Conner et al.,Proc. Natl. Acad. Sci. USA 80: 278 (1983), oligonucleotide ligationassays (OLAs) (Landegren et al., Science 241: 1077 (1988)), RNAseProtection Assay and the like. Molecular techniques for DNA analysishave been reviewed (Landegren et al, Science, 242: 229-237 (1988)).Following DNA amplification, the reaction product may be detected bySouthern blot analysis, without using radioactive probes. In such aprocess, for example, a small sample of DNA containing thepolynucleotides obtained from the tissue or subject are amplified, andanalyzed via a Southern blotting technique. The use of non-radioactiveprobes or labels is facilitated by the high level of the amplifiedsignal. In one embodiment of the invention, one nucleoside triphosphateis radioactively labeled, thereby allowing direct visualization of theamplification product by autoradiography. In another embodiment,amplification primers are fluorescently labeled and run through anelectrophoresis system. Visualization of amplified products is by laserdetection followed by computer assisted graphic display.

[0039] Simple visualization of a gel containing the separated productsmay be utilized to determine the presence or expression of PINCH. Forexample, staining of a gel to visualize separated polynucleotides, anumber of stains are well known to those skilled in the art. However,other is methods known to those skilled in the art may also be used, forexample scanning densitometry, computer aided scanning and quantitationas well as others.

[0040] Another technique which may be used to detect PINCH or ILKinvolves the use of antibodies. Such antibodies may consist of anantibody coupled to a low molecular weight hapten. These haptens canthen be specifically detected by means of a second reaction. Forexample, it is common to use such haptens as biotin, which reacts withavidin, or dinitrophenyl, pyridoxal, and fluorescein, which can reactwith specific antihapten antibodies.

[0041] The method for detecting a cell expressing PINCH or a cellproliferative disorder associated with PINCH, described above, can beutilized for detection of breast cancer or other malignancies in asubject, including those in a state of clinical remission. Additionally,the method for detecting PINCH polypeptide in cells is useful fordetecting a cell proliferative disorder by measuring the level of PINCHin cells or in a suspect tissue in comparison with PINCH expressed in anormal or standard cell or tissue. Using the method of the invention,PINCH expression can be identified in a cell and the appropriate courseof treatment can be employed (e.g., antisense gene therapy or drugtherapy). The expression pattern of PINCH may vary with the stage ofmalignancy of a cell, therefore, a sample such as breast tissue can bescreened with a panel of PINCH-specific reagents (i.e., nucleic acidprobes or antibodies to PINCH) to detect PINCH expression and diagnosethe stage of malignancy of the cell. Additionally, the inventors havediscovered that expression of ILK is related to cell proliferativedisorders. Thus, as discussed more fully below, expression of ILK isindicative of a metastatic cell proliferative disorder. In this regard,the sample may also be screened with polynucleotides to ILK orantibodies to an ILK polypeptide, wherein detection of over-expressedILK and PINCH would be indicative of a metastatic cell proliferativedisorder. Polynucleotide probes to ILK can be developed using techniqueswell known in the art. For example, a complementary polynucloeotidestrand to an ILK polynucleotide can be used as a probe in Northern andSouthern blots. Additionally, an oligonucleotide complementary to an ILKpolynucleotide can also be used as a probe or as a primer for PCRamplification of ILK. Finally, antibodies can be used to detectexpression of ILK polypeptides. The antibodies may be monoclonal orpolyclonal.

[0042] Monoclonal antibodies used in the method of the invention aresuited for use, for example, in immunoassays in which they can beutilized in liquid phase or bound to a solid phase carrier. In addition,the monoclonal antibodies in these immunoassays can be detectablylabeled in various ways. Examples of types of immunoassays which canutilize monoclonal antibodies of the invention are competitive andnon-competitive immunoassays in either a direct or indirect format.Examples of such immunoassays are the radioimmunoassay (RIA) and thesandwich (immunometric) assay. Detection of the antigens using themonoclonal antibodies of the invention can be done utilizingimmunoassays which are run in either the forward, reverse, orsimultaneous modes, including immunohistochemical assays onphysiological samples. Those of skill in the art will know, or canreadily discern, other immunoassay formats without undueexperimentation.

[0043] The term “immunometric assay” or “sandwich immunoassay”, includessimultaneous sandwich, forward sandwich and reverse sandwichimmunoassays. These terms are well understood by those skilled in theart. Those of skill will also appreciate that antibodies according tothe present invention will be useful in other variations and forms ofassays which are presently known or which may be developed in thefuture. These are intended to be included within the scope of thepresent invention.

[0044] Monoclonal antibodies can be bound to many different carriers andused to detect the presence of PINCH and/or ILK. Examples of well-knowncarriers include glass, polystyrene, polypropylene, polyethylene,dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, agaroses and magnetite. The nature of the carrier canbe either soluble or insoluble for purposes of the invention. Thoseskilled in the art will know of other suitable carriers for bindingmonoclonal antibodies, or will be able to ascertain such using routineexperimentation.

[0045] For purposes of the invention, PINCH and/or ILK may be detectedby the monoclonal antibodies when present in biological fluids andtissues. Any sample containing a detectable amount of PINCH or ILK canbe used. A sample can be a liquid such as blood, serum and the like, ora solid or semi-solid such as tissues, feces, and the like, or,alternatively, a solid tissue such as those commonly used inhistological diagnosis.

[0046] In performing the assays it may be desirable to include certain“blockers” in the incubation medium (usually added with the labeledsoluble antibody). The “blockers” are added to assure that non-specificproteins, proteases, or anti-heterophilic immunoglobulins to anti-PINCHimmunoglobulins present in the experimental sample do not cross-link ordestroy the antibodies on the solid phase support, or the radiolabeledindicator antibody, to yield false positive or false negative results.The selection of “blockers” therefore may add substantially to thespecificity of the assays described in the present invention.

[0047] It has been found that a number of nonrelevant (i.e.,nonspecific) antibodies of the same class or subclass (isotype) as thoseused in the assays (e.g., IgG1, IgG2a, IgM, etc.) can be used as“blockers”. The concentration of the “blockers” (normally 1-100 μg/μl)may be important, in order to maintain the proper sensitivity yetinhibit any unwanted interference by mutually occurring cross reactiveproteins in the specimen.

[0048] In vivo Diagnostics

[0049] Monoclonal antibodies may be used for the in vivo detection ofantigen, the detectably labeled monoclonal antibody is given in a dosewhich is diagnostically effective. The term “diagnostically effective”means that the amount of detectably labeled monoclonal antibody isadministered in sufficient quantity to enable detection of the sitehaving the PINCH or ILK antigen for which the monoclonal antibodies arespecific.

[0050] The concentration of detectably labeled monoclonal antibody whichis administered should be sufficient such that the binding to thosecells having PINCH or ILK is detectable compared to the background.Further, it is desirable that the detectably labeled monoclonal antibodybe rapidly cleared from the circulatory system in order to give the besttarget-to-background signal ratio. typically, the monoclonal antibody isreadioactively labelled, however, other labels are within the scope ofthe invention, for example, paramagnetic isotopes.

[0051] Additionally, monoclonal antibodies to ILK may be administeredalone or simultaneously with PINCH.

[0052] As a general rule, the dosage of detectably labeled monoclonalantibody for in vivo diagnosis will vary depending on such factors asage, sex, and extent of disease of the individual. The dosage ofmonoclonal antibody can vary from about 0.001 mg/m² to about 500 mg/m²,preferably 0.1 mg/m² to about 200 mg/m², most preferably about 0.1 mg/m²to about 10 mg/m². Such dosages may vary, for example, depending onwhether multiple injections are given, tumor burden, and other factorsknown to those of skill in the art.

[0053] For in vivo diagnostic imaging, the type of detection instrumentavailable is a major factor in selecting a given radioisotope. Theradioisotope chosen must have a type of decay which is detectable for agiven type of instrument. Still another important factor in selecting aradioisotope for in vivo diagnosis is that the half-life of theradioisotope be long enough so that it is still detectable at the timeof maximum uptake by the target, but short enough so that deleteriousradiation with respect to the host is minimized. Ideally, a radioisotopeused for in vivo imaging will lack a particle emission, but produce alarge number of photons in the 140-250 keV range, which may be readilydetected by conventional gamma cameras.

[0054] For in vivo diagnosis, radioisotopes may be bound toimmunoglobulin either directly or indirectly by using an intermediatefunctional group. Intermediate functional groups which often are used tobind radioisotopes which exist as metallic ions to immunoglobulins arethe bifunctional chelating agents such as diethylenetriaminepentaceticacid (DTPA) and ethylenediaminetetraacetic acid (EDTA) and similarmolecules. Typical examples of metallic ions which can be bound to themonoclonal antibodies of the invention are ¹¹¹In, ⁹⁷Ru, ⁶⁷Ga, ⁶⁸Ga,⁷²As, ⁸⁹Zr, and ²⁰¹Tl.

[0055] A monoclonal antibody useful in the method of the invention canalso be labeled with a paramagnetic isotope for purposes of in vivodiagnosis, as in magnetic resonance imaging (MRI) or electron spinresonance (ESR). In general, any conventional method for visualizingdiagnostic imaging can be utilized. Usually gamma and positron emittingradioisotopes are used for camera imaging and paramagnetic isotopes forMRI. Elements which are particularly useful in such techniques include¹⁵⁷Gd, ⁵⁵M, ¹⁶²Dy, ⁵²Cr, and ⁵⁶Fe.

[0056] Monoclonal antibodies used in the method of the invention can beused to monitor the course of amelioration of PINCH associated cellproliferative disorder. Thus, by measuring the increase or decrease inthe number of cells expressing PINCH or changes in PINCH present invarious body fluids, such as blood or serum, it would be possible todetermine whether a particular therapeutic regiment aimed atameliorating the disorder is effective.

[0057] Therapeutic Techniques

[0058] The present invention also provides a method for treating asubject with PINCH-associated cell proliferative disorder. For example,in breast cancer, the PINCH nucleotide sequence is over-expressed in acell as compared to expression in a normal-standard cell, therefore, itis possible to design appropriate therapeutic or diagnostic techniquesdirected to this sequence. Thus, where a cell-proliferative disorder isassociated with the expression of PINCH, polynucleotide sequences thatmodulate PINCH expression at the transcriptional or translational levelcan be used. In cases when a cell proliferative disorder or abnormalcell phenotype is associated with the under expression of PINCH, forexample, nucleic acid sequences encoding PINCH (sense) could beadministered to the subject with the disorder. In cases when a cellproliferative disorder, such as those seen in a breast caner, isassociated with the over-expression of PINCH an antisense or ribozymetherapy may be appropriate.

[0059] The term “cell-proliferative disorder” denotes malignant as wellas non-malignant cell populations which often appear to differ from thesurrounding tissue both morphologically and genotypically. Suchdisorders may be associated, for example, with over-expression of PINCH,ILK or a combination thereof. Essentially, any disorder which isetiologically linked to expression of PINCH could be consideredsusceptible to treatment with a reagent of the invention which modulatesPINCH expression.

[0060] The term “modulate” or “regulates” envisions the augmentation ofPINCH activity or PINCH gene expression. When a cell proliferativedisorder is associated with PINCH expression, antibodies which bindPINCH may prevent PINCH activity. Similarly, antisense oligonucleotidesmay bind to DNA or RNA encoding PINCH and thus prevent its expression.

[0061] Antisense oligonucleotides can effectively reduce PINCHexpression and can be used to treat disease associated with PINCH, suchas signal transduction disorders, including cell proliferativedisorders, neoplasms, or cancer, for example breast cancer. Theantisense oligonucleotides can be delivered to cells in culture or tocells or tissues in humans or delivered in animal models having thesediseases. Binding of PINCH polynucleotide by an antisenseoligonucleotide can be used to inhibit cell proliferation associatedwith cell proliferative disorders. Furthermore, when used in combinationwith antisense oligonucleotides to ILK, the combination therapy can beused to inhibit metastatic cell proliferative disorders.

[0062] “Antisense oligonucleotide” means any RNA or DNA molecules whichcan bind specifically with a targeted polynucleotide sequence,interrupting the expression of that gene's protein product. Theantisense molecule binds to either the messenger RNA forming a doublestranded molecule which cannot be translated by the cell or to the DNAor other polynucleotide encoding PINCH or ILK. Antisenseoligonucleotides of about 8 to 40 nucleic acids and more preferablyabout 13-30 are preferred since they are easily synthesized and have aninhibitory effect just like antisense RNA molecules. In addition,chemically reactive groups, such as iron-linkedethylenediaminetetraacetic acid (EDTA-Fe) can be attached to anantisense oligonucleotide, causing cleavage of the RNA at the site ofhybridization. These and other uses of antisense methods to inhibit thein vitro translation of genes are well known in the art (Marcus-Sakura,1988, Anal., Biochem., 172:289).

[0063] Antisense oligonucleotides are DNA or RNA molecules that arecomplementary to, at least a portion of, a specific polynucleotidemolecule (Weintraub, Scientific American, 262:40, 1990). In the cell,the antisense oligonucleotides hybridize to the corresponding targetpolynucleotide, forming a double-stranded or triplex molecule. Theantisense oligonucleotides interfere with the translation of, forexample, mRNA, since the cell will not translate a mRNA that isdouble-stranded. Antisense oligomers of about 8 to 40 nucleotides arepreferred, since they are easily synthesized and are less likely tocause problems than larger molecules when introduced into the targetPINCH producing cell.

[0064] Use of a oligonucleotides to stall transcription is known as thetriplex strategy since the oligomer winds around double-helical DNA,forming a three-strand helix. Therefore, these triplex compounds can bedesigned to recognize a unique site on a chosen gene (Maher, et al.,1991, Antisense Res. and Dev., 1(3):227; Helene, C., 1991, AnticancerDrug Design, 6(6 :569).

[0065] These and other uses of antisense methods to inhibit the in vivotranscription or translation of genes are well known in the art (e.g.,De Mesmaeker, et al., 1995. Backbone modifications in oligonucleotidesand peptide nucleic acid systems. Curr. Opin. Struct. Biol. 5:343-355;Gewirtz, A. M., et al., 1996b. Facilitating delivery of antisenseoligodeoxynucleotides: Helping antisense deliver on its promise; Proc.Natl. Acad. Sci. U.S.A. 93:3161-3163; Stein, C. A. A discussion ofG-tetrads 1996. Exploiting the potential of antisense: beyondphosphorothioate oligodeoxynucleotides. Chem. and Biol. 3:319-323).

[0066] The invention includes antisense oligonucleotides which hybridizewith a polynucleotide sequence comprising SEQ ID NO:1 or its complement.The antisense oligonucleotides employed may be unmodified or modifiedRNA or DNA molecules. Suitable modifications include, but are notlimited to, the ethyl or methyl phosphorate modification disclosed inU.S. Pat. No. 4,469,863, the disclosure of which is incorporated byreference, and the phosphorthioate modifications to deoxynucleotidesdescribed by LaPlanche, et al., 1986 Nucleic Acids Research, 14:9081,and by Stec, et al., 1984 J Am. Chem Soc. 106:6077. The modification tothe antisense oligonucleotides is preferably a terminal modification inthe 5′ or 3′ region. Preferred are modifications of the 3′ terminalregion. Also preferred are modifications with methyl groups added to 5′carbon atoms as described by Saha, et al., 1993 CEN, 44:44.

[0067] Phosphodiester-linked oligonucleotides are particularlysusceptible to the action of nucleases in serum or inside cells, andtherefore in a preferred embodiment the antisense molecules of thepresent invention are phosphorothioate or methyl phosphonate-linkedanalogues, which have been shown to be nuclease-resistant. Specificexamples of some preferred oligonucleotides envisioned for thisinvention may contain phosphorothioates, phosphotriesters, methylphosphonates, short chain alkyl or cycloalkyl intersugar linkages orshort chain heteroatomic or heterocyclic intersugar (“backbone”)linkages. Most preferred are phosphorothioates and those with CH₂NHOCH₂,CH₂N(CH₃)OCH₂, CH₂ON(C₄)CH₂, CH₂N(CH₃)N(CH₃)CH₃ and ON(CH₃)CH₂CH₂backbones (where phosphodiester is OPOCH₂). Also preferred areoligonucleotides having morpholino backbone structures (Summerton, J. E.and Weller, D. D., U.S. Pat. No. 5,034,506). In other preferredembodiments, 2′-methylribonucleotides (Inoue, et al., 1987 Nucleic AcidsResearch, 15:6131) and chimeric oligonucleotides that are compositeRNA-DNA analogues (Inoue, et al., 1987 FEBS Lett., 215:327) may also beused for the purposes described herein. Finally, DNA analogues, such aspeptide nucleic acids (PNA) are also included (Egholm, et al., 1993Nature 365:566; P. E. Nielsen, M. Egholm, R. H. Berg, O. Buchardt, 1991Science, 254:1497) and can be used according to the invention. Otherpreferred oligonucleotides may contain alkyl and halogen-substitutedsugar moieties comprising one of the following at the 2′ position: OH,SH, SCH₃, F, OCN, OCH₃OCH₃, OCH₃O(CH₂)nCH₃, O(CH₂)nNH₂ or O(CH₂)nCH₃where n is from 1 to about 10; C1 to C10 lower alkyl, substituted loweralkyl, alkaryl or aralkyl; Cl; Br; CN; CF₃; OCF₃; O, S , or N-alkyl; O,S or N alkenyl; SOCH₃; SO₂CH; ON₂O; N₂O; N₃; N₃H; heterocycloalkyl;heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl;an RNA cleaving group; a cholesteryl group; a conjugate; a reportergroup; an intercalator; a group for improving the pharmacokineticproperties of a oligonucleotide; or a group for improving thepharmacodynamic properties of a oligonucleotide and other substituentshaving similar properties. Oligonucleotides may also have sugar mimeticssuch as cyclobutyls in place of the pentofuranosyl group. Otherpreferred embodiments may include at least one modified base form or“universal base” such as inosine. The preparation of base-modifiednucleosides, and the synthesis of modified oligonucleotides using saidbase-modified nucleosides as precursors, has been described, forexample, in U.S. Pat. Nos. 4,948,882 and 5,093,232. These base-modifiednucleosides have been designed so that they can be incorporated bychemical synthesis into either terminal or internal positions of aoligonucleotide. Such base-modified nucleosides, present at eitherterminal or internal positions of a oligonucleotide, can serve as sitesfor attachment of a peptide or other antigen. Nucleosides modified intheir sugar moiety have also been described (e.g., U.S. Pat. No.5,118,802) and can be used similarly. Persons of ordinary skill in thisart will be able to select other linkages for use in the invention.These modifications also may be designed to improve the cellular uptakeand stability of the oligonucleotides. It is understood that dependingon the route or form of administration of the antisense oligonucleotidesof the invention, the modification or site of modification will vary(e.g., 5′ or 3′ modification). One of skill in the art could readilydetermine the appropriate modification without undue experimentation.

[0068] In order for the target cell, tissue or subject to be renderedsusceptible to the antisense oligonucleotides in accordance with themethod of the invention, the cells must be exposed to theoligonucleotide under condition that facilitate their uptake by thecell, tissue or subject. In vitro therapy may be accomplished by anumber of procedures, including, for example, simple incubation of thecells or tissue with the oligonucleotide in a suitable nutrient mediumfor a period of time suitable to inhibit PINCH production.

[0069] The antisense oligonucleotides of the invention can be deliveredalone or in conjunction with other agents such as immunosuppressivedrugs, ribozymes or other antisense molecules. For example, ribozymes orantisense molecules that specifically bind mRNA encoding ILK, or acytokine, such as TNF-α or interferon-γ, can be used with the antisensemolecules of the present invention.

[0070] Additionally, the antisense oligonucleotides of the presentinvention may be administered ex vivo by harvesting cells or tissue froma subject, treating them with the antisense oligonucleotide, thenreturning the treated cells or tissue to the subject. The presentinvention provides method for the treatment of a disease which isassociated with PINCH. Such therapy would achieve its therapeutic effectby introduction of the appropriate antisense oligonucleotide which bindspolynucleotides encoding PINCH into cells of subjects having thedisorder. Delivery of the PINCH antisense molecule can be achieved usinga recombinant expression vector such as a chimeric virus or a colloidaldispersion system.

[0071] Many of the methods as described herein can be performed in vivoor ex vivo. Various viral vectors which can be utilized for gene therapyas taught herein include adenovirus, herpes virus, vaccinia, or,preferably, an RNA virus such as a retrovirus. Preferably, theretroviral vector is a derivative of a murine or avian retrovirus.Examples of retroviral vectors in which a single foreign gene can beinserted include, but are not limited to: Moloney murine leukemia virus(MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumorvirus (MuMTV), and Rous Sarcoma Virus (RSV). Preferably, when thesubject is a human, a vector such as the gibbon ape leukemia virus(GaLV) is utilized. A number of additional retroviral vectors canincorporate multiple genes. All of these vectors can transfer orincorporate a gene for a selectable marker so that transduced cells canbe identified and generated. By inserting a sequence encoding anantisense oligonucleotide which specifically binds polynucleotidesencoding PINCH into the viral vector, along with another gene whichencodes the ligand for a receptor on a specific target cell, forexample, the vector is now target specific. Preferred targeting isaccomplished by using an antibody to target the retroviral vector. Thoseof skill in the art will know of, or can readily ascertain without undueexperimentation, specific polynucleotide sequences which can be insertedinto the retroviral genome, for example, to allow target specificdelivery of the retroviral vector containing the antisenseoligonucleotide.

[0072] Since recombinant retroviruses are defective, they requireassistance in order to produce infectious vector particles. Thisassistance can be provided, for example, by using helper cell lines thatcontain plasmids encoding all of the structural genes of the retrovirusunder the control of regulatory sequences within the LTR. These plasmidsare missing a nucleotide sequence which enables the packaging mechanismto recognize an RNA transcript for encapsidation. Helper cell lineswhich have deletions of the packaging signal include but are not limitedto Ψ2, PA317 and PA12, for example. These cell lines produce emptyvirions, since no genome is packaged. If a retroviral vector isintroduced into such cells in which the packaging signal is intact, butthe structural genes are replaced by other genes of interest, the vectorcan be packaged and vector virion produced.

[0073] Alternatively, NIH 3T3 or other tissue culture cells can bedirectly transfected with plasmids encoding the retroviral structuralgenes gag, pol and env, by conventional calcium phosphate transfection.These cells are then transfected with the vector plasmid containing thegenes of interest. The resulting cells release the retroviral vectorinto the culture medium.

[0074] Another targeted delivery system for antisense oligonucleotidesthat bind polynucleotides encoding PINCH is a colloidal dispersionsystem. Colloidal dispersion systems include macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Thepreferred colloidal system of this invention is a liposome. Liposomesare artificial membrane vesicles which are useful as delivery vehiclesin vitro and in vivo. It has been shown that large unilamellar vesicles(LUV), which range in size from 0.2-4.0 μm can encapsulate a substantialpercentage of an aqueous buffer containing large macromolecules. RNA,DNA and intact virions can be encapsulated within the aqueous interiorand be delivered to cells in a biologically active form (Fraley, et al.,1981 Trends Biochem. Sci., 6:77). In order for a liposome to be anefficient gene transfer vehicle, the following characteristics should bepresent: (1) encapsulation of the genes of interest at high efficiencywhile not compromising their biological activity; (2) preferential andsubstantial binding to a target cell in comparison to non-target cells;(3) delivery of the aqueous contents of the vesicle to the target cellcytoplasm at high efficiency; and (4) accurate and effective expressionof genetic information (Mannino, et al., 1988 Biotechniques, 6:682).

[0075] The composition of the liposome is usually a combination ofphospholipids, particularly high-phase-transition-temperaturephospholipids, usually in combination with steroids, especiallycholesterol. Other phospholipids or other lipids may also be used. Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations.

[0076] Examples of lipids useful in liposome production includephosphatidyl compounds, such as phosphatidylglycerol,phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,sphingolipids, cerebrosides, and gangliosides. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

[0077] The targeting of liposomes has been classified based onanatomical and mechanistic factors. Anatomical classification is basedon the level of selectivity, for example, organ-specific, cell-specific,and organelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

[0078] The surface of the targeted delivery system may be modified in avariety of ways. In the case of a liposomal targeted delivery system,lipid groups can be incorporated into the lipid bilayer of the liposomein order to maintain the targeting ligand in stable association with theliposomal bilayer. Various linking groups can be used for joining thelipid chains to the targeting ligand. In general, the compounds bound tothe surface of the targeted delivery system will be ligands andreceptors which will allow the targeted delivery system to find and“home in” on the desired cells. A ligand may be any compound of interestwhich will bind to another compound, such as a receptor.

[0079] Another delivery system for the antisense oligonucleotides of theinvention at particular sites in a subject, includes the use ofgene-activated matrices. In this system the antisense molecule is coatedon a biocompatible matrix, sponge or scaffold and implanted at thetissue site wherein cells proliferate and grow on the scaffold, takingup the antisense oligonucleotide (See for example U.S. Pat. No.5,763,416, which is incorporated herein by reference).

[0080] In yet another delivery system, the antisense molecules of theinvention may be microinjected into cells. The antisense molecules maybe prepared in an appropriate buffer and the naked oligonucleotide,either alone or contained in an appropriate vector, microinjected, forexample, into a stem cell of a tissue to be treated.

[0081] In addition, antisense oligonucleotides according to theinvention may also be administered in vivo. Antisense oligonucleotidescan be administered as a compound or as a pharmaceutically acceptablesalt of the compound, alone or in combination with pharmaceuticallyacceptable carriers, diluents, simple buffers, and vehicles. Forexample, expression vectors that produce antisense molecules can beengineered from DNA duplexes in the laboratory and introduced into cells(Weintraub, et al., 1990 Sci. Amer. 1:40). Most preferably, antisenseoligonucleotides are mixed individually or in combination withpharmaceutically acceptable carriers to form compositions which allowfor easy dosage preparation.

[0082] An antisense oligonucleotide of the invention can be administeredto provide in vivo therapy to a subject having a disorder which isassociate with PINCH expression. Such therapy can be accomplished byadministering ex vivo and in vivo as the case may be, a therapeuticallyeffective amount of antisense oligonucleotide. The term “therapeuticallyeffective” means that the amount of antisense oligonucleotideadministered is of sufficient quantity to suppress, to some beneficialdegree, expression of PINCH.

[0083] Antisense oligonucleotide according to the present invention canbe administered to the patient in any acceptable manner includingorally, by injection, using an implant, nasally and the like. Oraladministration includes administering an oligonucleotide of the presentinvention in tablets, suspension, implants, solutions, emulsions,capsules, powders, syrups, water composition, and the like. Nasaladministration includes administering the composition of the presentinvention in sprays, solutions and the like. Injections and implants arepreferred because they permit precise control of the timing and dosagelevels useful for administration, with injections being most preferred.Antisense oligonucleotides are preferably administered parenterally.

[0084] The therapeutic agents useful in the method of the invention canbe administered parenterally by injection or by gradual perfusion overtime. Administration may be intravenously, intra-peritoneally,intramuscularly, subcutaneously, intra-cavity, or transdermally.

[0085] Preparations for parenteral administration include sterileaqueous or non-aqueous solutions, suspensions, and emulsions. Examplesof non-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's intravenousvehicles include fluid and nutrient replenishers, electrolytereplenishers (such as those based on Ringer's dextrose), and the like.Preservatives and other additives may also be present such as, forexample, antimicrobials, anti-oxidants, chelating agents and inert gasesand the like.

[0086] The invention also includes a composition for therapy comprisingan effective amount of an enzymatic RNA of the invention or combinationthereof, and a physiologically acceptable excipient or carrier.

[0087] Physiologically acceptable and pharmaceutically acceptableexcipients and carriers are well known to those of skill in the art. By“physiologically or pharmaceutically acceptable carrier” as used hereinis meant any substantially non-toxic carrier for administration in whichan antisense oligonucleotide of the invention will remain stable andbioavailable when used. For example, the antisense oligonucleotide ofthe invention can be dissolved in a liquid, dispersed or emulsified in amedium in a conventional manner to form a liquid preparation or is mixedwith a semi-solid (gel) or solid carrier to form a paste, ointment,cream, lotion or the like.

[0088] Suitable carriers include water, petroleum jelly (vaseline),petrolatum, mineral oil, vegetable oil, animal oil, organic andinorganic waxes, such as microcrystalline, paraffin and ozocerite wax,natural polymers, such as xanthanes, gelatin, cellulose, or gum arabic,synthetic polymers, such as discussed below, alcohols, polyols, waterand the like. Preferably, because of its non-toxic properties, thecarrier is a water miscible carrier composition that is substantiallymiscible in water. Such water miscible carrier composition can includethose made with one or more ingredients set forth above but can alsoinclude sustained or delayed release carrier, including watercontaining, water dispersable or water soluble compositions, such asliposomes, microsponges, microspheres or microcapsules, aqueous baseointments, water-in-oil or oil-in-water emulsions or gels.

[0089] The carrier can comprise a sustained release or delayed releasecarrier. The carrier is any material capable of sustained or delayedrelease of the antisense molecule specifically directed against PINCHpolynucleotide to provide a more efficient administration resulting inone or more of less frequent and/or decreased dosage of the antisensemolecule, ease of handling, and extended or delayed effects. The carrieris capable of releasing the oligomer when exposed to the environment ofthe area for diagnosis or treatment or by diffusing or by releasedependent on the degree of loading of the oligonucleotide to the carrierin order to obtain release of the antisense oligonucleotide of theinvention. Non-limiting examples of such carriers include liposomes,microsponges, microspheres, gene-activated matrices, as described above,or microcapsules of natural and synthetic polymers and the like.Examples of suitable carriers for sustained or delayed release in amoist environment include gelatin, gum arabic, xanthane polymers; bydegree of loading include lignin polymers and the like; by oily, fattyor waxy environment include thermoplastic or flexible thermoset resin orelastomer including thermoplastic resins such as polyvinyl halides,polyvinyl esters, polyvinylidene halides and halogenated polyolefins,elastomers such as brasiliensis, polydienes, and halogenated natural andsynthetic rubbers, and flexible thermoset resins such as polyurethanes,epoxy resins and the like.

[0090] Preferably, the sustained or delayed release carrier is aliposome, microsponge, microsphere or gel.

[0091] The compositions of the invention are administered by anysuitable means, including injection, implantation, transdermal,intraocular, transmucosal, bucal, intrapulmonary, and oral.

[0092] Preferably the carrier is a pH balanced buffered aqueous solutionfor injection. However, the preferred carrier will vary with the mode ofadministration.

[0093] The compositions for administration usually contain from about0.0001% to about 90% by weight of the antisense oligonucleotide of theinvention compared to the total weight of the composition, preferablyfrom about 0.5% to about 20% by weight of the antisense oligonucleotideof the invention compared to the total composition, and especially fromabout 2% to about 20% by weight of the antisense oligonucleotide of theinvention compared to the total composition.

[0094] The effective amount of the antisense oligonucleotide of theinvention used for therapy or diagnosis of course can vary depending onone or more of factors such as the age and weight of the patient, thetype of formulation and carrier ingredients, frequency of use, the typeof therapy or diagnosis preformed and the like. It is a simple matterfor those of skill in the art to determine the precise amounts to usetaking into consideration these factors and the present specification.

[0095] The surface of the targeted delivery system may be modified in avariety of ways. In the case of a liposomal targeted delivery system,lipid groups can be incorporated into the lipid bilayer of the liposomein order to maintain the targeting ligand in stable association with theliposomal bilayer. Various linking groups can be used for joining thelipid chains to the targeting ligand.

[0096] In general, the compounds bound to the surface of the targeteddelivery system will be ligands and receptors which will allow thetargeted delivery system to find and “home in” on the desired cells. Aligand may be any compound of interest which will bind to anothercompound, such as a receptor.

[0097] In general, surface membrane proteins which bind to specificeffector molecules are referred to as receptors. In the presentinvention, antibodies are preferred receptors. Antibodies can be used totarget liposomes to specific cell-surface ligands. For example, certainantigens expressed specifically on tumor cells, referred to astumor-associated antigens (TAAs), may be exploited for the purpose oftargeting PINCH antibody-containing liposomes directly to the malignanttumor. Since the PINCH gene product may be indiscriminate with respectto cell type in its action, a targeted delivery system offers asignificant improvement over randomly injecting non-specific liposomes.Preferably, the target tissue is a breast tissue. A number of procedurescan be used to covalently attach either polyclonal or monoclonalantibodies to a liposome bilayer. Antibody-targeted liposomes caninclude monoclonal or polyclonal antibodies or fragments thereof such asFab, or F(ab′)₂, as long as they bind efficiently to an the antigenicepitope on the target cells. Liposomes may also be targeted to cellsexpressing receptors for hormones or other serum factors.

[0098] Pinch Regulating and Blocking Agents

[0099] In yet another embodiment, the present invention relates toPINCH-binding and/or blocking agents. Such agents could representresearch and diagnostic tools in the study of cell proliferativedisorders. In addition, pharmaceutical compositions comprising isolatedand purified PINCH-binding agents may represent effective cancertherapeutics. The phrase “PINCH-binding agent” denotes an agents whichfunctions to bind PINCH polypeptide or the promotor region of the PINCHgene. The term includes both biologic agents and chemical compounds. Thedetermination and isolation of ligand/compositions is well described inthe art. See, e.g., Lerner, Trends NeuroSci. 17: 142-146 (1994), whichis hereby incorporated in its entirety by reference.

[0100] Screening for Pinch Binding Compounds

[0101] Also included is a method of identifying compounds that bindPINCH polypeptide or fragments thereof. The method for identifying acompound which binds to PINCH polypeptide comprises incubating a testcompound and PINCH polypeptide under conditions sufficient to allow thecompound and PINCH polypeptide to form a complex. Conditions will varyaccording to a number of factors well recognized in the art, includingtemperature, concentration, hydrophobicity and hydrophilicity of themolecules, for example hydrophobic molecules such as long chain fattyacids may require buffers including dimethylsulfoxide (DMSO). Compoundscan thus be any number of molecules including polypeptides,peptidomimetics, carbohydrates, fatty acids, and/or steroids. Themolecules may be labeled. For example the PINCH or the test compound maybe chemically labeled with a fluorescence compound, a radioactiveelement or a metal chelating agent. The method also involves separatinga complex of PINCH polypeptide and the binding compound from unboundPINCH polypeptide and measuring the binding or effect of binding of thecompound to PINCH polypeptide. Separation of the compounds and PINCH canbe accomplished by any number of means including chromatography, gelelectrophoresis and other well known to those skilled in the art.

EXAMPLE

[0102] Antibodies and anti-sera were prepared by creating a 6-histidinePINCH fusion protein by ligation of a PCR product containing the entirehuman PINCH open reading frame in-frame with the pAcSG His NTbaculovirus transfer vector (PharMingen), which was then co-transfectedwith BACULOGOLD DNA (a modified baculovirus DNA) into insect cells.Recombinant PINCH was purified by chromatography of insect cell lysateson a metal chelate matrix (ProBond, Invitrogen). Rabbits were immunizedwith recombinant PINCH isolated from SDS-polyacrylamide gels, andantisera produced at Rackland Laboratories, Gilbertsville, Pa. Rabbitanti-PINCH IgG was affinity-purified using a GST fusion proteincorresponding to the third PINCH LIM domain and used at 1 ug/ml. Rabbitanti-ILK (Upstate Biotechnology) was used at 1 ug/ml, mouse anti-cylniD1 (Santa Cruz) was used at 0.2 ug/ml, and mouse anti-erbB-2(NovoCastra) was used at 1:500.

[0103] To determine whether PINCH and ILK had affects in cellproliferative disorders associated with cell signaling, excesspathological material was obtained from the Clinical Laboratory of theUniversity of Calif., San Diego Medical Center-Thorton Hospitalaccording to guidelines established by the institutional reviewcommittee. The tissues were solubilized in RIPA buffer (50 mM sodiumborate, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate)containing protease inhibitors (0.1 mg/ml phenylmethylsulfonyl fluoride,1 ug/ml aprotinin and 1 ug/ml leupeptin). Protein concentration of thetissue lysates were determined by the dotMETRIC assay (ChemiconInternational) following the manufacturer's instructions and confirmedby Comassie blue staining of SDS-polyacrylamide gels.

[0104] Solubilized breast proteins were boiled in loading buffercontaining 200 mM dithiothreitol and 720 mM 2-mercaptoethanol for 5minutes, separated by electrophoresis on SDS-polyacrylamide gels andtransferred to nitrocellulose (Hybond-ECL, Amersham) in transfer buffer(25 mM ethanolamine/glycine, pH 9.5, containing 20% methanol).Immunoblots were blocked with 5% blocking agent (Amersham) inTris-buffered saline, pH 7.5 (TBS) and reacted overnight at 4° C. withantibody in TBS with 1% blocking agent. Following washing in TBS/0.1%Tween-20, immunoblots were reacted for 30 minutes with the appropriatehorseradish peroxidase-conjugated anti-rabbit or anti-mouse Ig(Amersham). Reactions were detected by enhanced chemiluminescence (ECLdetection kit, Amersham). To estimate the magnitude of specific proteinincreases in breast cancer, a known quantity of normal breast tissuelysate was compared on immunoblots to the same quantity of breast cancertissue lysate, as well as to {fraction (1/3)} and {fraction (1/6)}quantities of breast cancer tissue lysate. Anti-cyclin D1 stainingshowed high sensitivity, detecting cyclin D1 in all samples includingthe normals, and therefore cyclin D1 was determined to be increased whenit could clearly be detected at {fraction (1/3)} quantity.

[0105] Using the techniques described, the cases examined represented aspectrum of breast cancer diagnoses, and varied in expression of knownprognostic indicators: {fraction (4/6)} breast cancer cases werepositive for cyclin D1, {fraction (3/6)} were positive for erbB-2(HER2/neu), and {fraction (2/6)} were positive for estrogen andprogesterone receptors (FIG. 1). The greatest increase in PINCH and ILKwas found in cases of metastatic breast carcinoma (case 7, FIG. 1) whichwas positive for cyclin D1 and erbB-2 and negative for estrogen andprogesterone receptors. The most modest increase in PINCH and ILK wasfound in a low-risk breast cancer case (case 3, FIG. 1) which incontrast to case 7, was negative for cyclin D1 and erbB-2 and positivefor estrogen and progesterone receptors. Thus, the study demonstratesthat the adapter protein PINCH and its associated serine-threoninekinase ILK are significantly increased over normal in a sample of breastcancer tissue lysates. All six breast cancer tissue lysates showedincreased PINCH and ILK, suggesting that upregulation of the PINCHsignaling complex may be an early event in breast cancer. Furthermore,case #3 was a small, localized lesion that was shown by DNA analysis tobe euploid and to have a low S phase fraction, while case #7 was anaggressive metastatic lesion. These results support the finding that ILKoverexpression is important in determining cell phenotype, and that thehigher the level o f ILK overexpression, the more aggressive andinvasive the tumor.

[0106] A number of embodiments of the present invention have beendescribed. Nevertheless, it will be understood that variousmodifications may be made without departing from the spirit and scope ofthe invention. Accordingly, other embodiments are within the scope ofthe following claims.

1 2 1 1246 DNA Homo sapiens CDS (120)...(1061) 1 tagttcaaga caacagagacaaagctaaga tgaggaagtt ctgtacagtt taggaaatag 60 aggctttcaa agataattcgcagtgatgtg aaactggcct cccaagccct gataacaac 119 atg gcc aac gcc ctg gccagc gcc act tgc gag cgc tgc aag ggc ggc 167 Met Ala Asn Ala Leu Ala SerAla Thr Cys Glu Arg Cys Lys Gly Gly 1 5 10 15 ttt gcg ccc gct gag aagatc gtg aac agt aat ggg gag ctg tac cat 215 Phe Ala Pro Ala Glu Lys IleVal Asn Ser Asn Gly Glu Leu Tyr His 20 25 30 gag cag tgt ttc gtg tgc gctcag tgc ttc cag cag ttc cca gaa gga 263 Glu Gln Cys Phe Val Cys Ala GlnCys Phe Gln Gln Phe Pro Glu Gly 35 40 45 ctc ttc tat gag ttt gaa gga agaaag tac tgt gaa cat gac ttt cag 311 Leu Phe Tyr Glu Phe Glu Gly Arg LysTyr Cys Glu His Asp Phe Gln 50 55 60 atg ctc ttt gcc cct tgc tgt cat cagtgt ggt gaa ttc atc att ggc 359 Met Leu Phe Ala Pro Cys Cys His Gln CysGly Glu Phe Ile Ile Gly 65 70 75 80 cga gtt atc aaa gcc atg aat aac agctgg cat ccg gag tgc ttc cgc 407 Arg Val Ile Lys Ala Met Asn Asn Ser TrpHis Pro Glu Cys Phe Arg 85 90 95 tgt gac ctc tgc cag gaa gtt ctg gca gatatc ggg ttt gtc aag aat 455 Cys Asp Leu Cys Gln Glu Val Leu Ala Asp IleGly Phe Val Lys Asn 100 105 110 gct ggg aga cac ctg tgt cgc ccc tgt cataat cgt gag aaa gcc aga 503 Ala Gly Arg His Leu Cys Arg Pro Cys His AsnArg Glu Lys Ala Arg 115 120 125 ggc ctt ggg aaa tac atc tgc cag aaa tgccat gct atc atc gat gag 551 Gly Leu Gly Lys Tyr Ile Cys Gln Lys Cys HisAla Ile Ile Asp Glu 130 135 140 cag cct ctg ata ttc aag aac gac ccc taccat cca gac cat ttc aac 599 Gln Pro Leu Ile Phe Lys Asn Asp Pro Tyr HisPro Asp His Phe Asn 145 150 155 160 tgc gcc aac tgc ggg aag gag ctg actgcc gat gca cgg gag ctg aaa 647 Cys Ala Asn Cys Gly Lys Glu Leu Thr AlaAsp Ala Arg Glu Leu Lys 165 170 175 ggg gag cta tac tgc ctc cca tgc catgat aaa atg ggg gtc ccc atc 695 Gly Glu Leu Tyr Cys Leu Pro Cys His AspLys Met Gly Val Pro Ile 180 185 190 tgt ggt gct tgc cga cgg ccc atc gaaggg cgc gtg gtg aac gct atg 743 Cys Gly Ala Cys Arg Arg Pro Ile Glu GlyArg Val Val Asn Ala Met 195 200 205 ggc aag cag tgg cat gtg gag cat tttgtt tgt gcc aag tgt gag aaa 791 Gly Lys Gln Trp His Val Glu His Phe ValCys Ala Lys Cys Glu Lys 210 215 220 ccc ttt ctt gga cat cgc cat tat gagagg aaa ggc ctg gca tat tgt 839 Pro Phe Leu Gly His Arg His Tyr Glu ArgLys Gly Leu Ala Tyr Cys 225 230 235 240 gaa act cac tat aac cag cta tttggt gat gtt tgc ttc cac tgc aat 887 Glu Thr His Tyr Asn Gln Leu Phe GlyAsp Val Cys Phe His Cys Asn 245 250 255 cgt gtt ata gaa ggt gat gtg gtctct gct ctt aat aag gcc tgg tgc 935 Arg Val Ile Glu Gly Asp Val Val SerAla Leu Asn Lys Ala Trp Cys 260 265 270 gtg aac tgc ttt gcc tgt tct acctgc aac act aaa tta aca ctc aag 983 Val Asn Cys Phe Ala Cys Ser Thr CysAsn Thr Lys Leu Thr Leu Lys 275 280 285 aat aag ttt gtg gag ttt gac atgaag cca gtc tgt aag aag tgc tat 1031 Asn Lys Phe Val Glu Phe Asp Met LysPro Val Cys Lys Lys Cys Tyr 290 295 300 gag att tcc att gga gct gaa gaaaag act taagaaacta gctgagacct 1081 Glu Ile Ser Ile Gly Ala Glu Glu LysThr 305 310 taggaaggaa ataagttcct ttattttttc ttttctatgc aagataagagattaccaaca 1141 ttacttgtct tgatctaccc atatttaaag ctatatctca aagcagttgagagaagagga 1201 cctatatgaa tggttttatg tcattttttt aaaaaaaaaa aaaaa 1246 2314 PRT Homo sapiens 2 Met Ala Asn Ala Leu Ala Ser Ala Thr Cys Glu ArgCys Lys Gly Gly 1 5 10 15 Phe Ala Pro Ala Glu Lys Ile Val Asn Ser AsnGly Glu Leu Tyr His 20 25 30 Glu Gln Cys Phe Val Cys Ala Gln Cys Phe GlnGln Phe Pro Glu Gly 35 40 45 Leu Phe Tyr Glu Phe Glu Gly Arg Lys Tyr CysGlu His Asp Phe Gln 50 55 60 Met Leu Phe Ala Pro Cys Cys His Gln Cys GlyGlu Phe Ile Ile Gly 65 70 75 80 Arg Val Ile Lys Ala Met Asn Asn Ser TrpHis Pro Glu Cys Phe Arg 85 90 95 Cys Asp Leu Cys Gln Glu Val Leu Ala AspIle Gly Phe Val Lys Asn 100 105 110 Ala Gly Arg His Leu Cys Arg Pro CysHis Asn Arg Glu Lys Ala Arg 115 120 125 Gly Leu Gly Lys Tyr Ile Cys GlnLys Cys His Ala Ile Ile Asp Glu 130 135 140 Gln Pro Leu Ile Phe Lys AsnAsp Pro Tyr His Pro Asp His Phe Asn 145 150 155 160 Cys Ala Asn Cys GlyLys Glu Leu Thr Ala Asp Ala Arg Glu Leu Lys 165 170 175 Gly Glu Leu TyrCys Leu Pro Cys His Asp Lys Met Gly Val Pro Ile 180 185 190 Cys Gly AlaCys Arg Arg Pro Ile Glu Gly Arg Val Val Asn Ala Met 195 200 205 Gly LysGln Trp His Val Glu His Phe Val Cys Ala Lys Cys Glu Lys 210 215 220 ProPhe Leu Gly His Arg His Tyr Glu Arg Lys Gly Leu Ala Tyr Cys 225 230 235240 Glu Thr His Tyr Asn Gln Leu Phe Gly Asp Val Cys Phe His Cys Asn 245250 255 Arg Val Ile Glu Gly Asp Val Val Ser Ala Leu Asn Lys Ala Trp Cys260 265 270 Val Asn Cys Phe Ala Cys Ser Thr Cys Asn Thr Lys Leu Thr LeuLys 275 280 285 Asn Lys Phe Val Glu Phe Asp Met Lys Pro Val Cys Lys LysCys Tyr 290 295 300 Glu Ile Ser Ile Gly Ala Glu Glu Lys Thr 305 310

What is claimed is:
 1. A method for identifying a compound whichmodulates cell proliferation, comprising contacting a sample containingPINCH polypeptide with a compound suspected of having PINCH modulatingactivity and detecting an effect on cell proliferation.
 2. The method ofclaim 1, wherein the compound is a peptide.
 3. The method of claim 1,wherein the compound is a peptidomimetic.
 4. The method of claim 1,wherein the sample comprising PINCH is expressed in a cell.
 5. Themethod of claim 1, wherein the compound is an antibody.